Method of producing cecropins by microbiological techniques

ABSTRACT

A polynucleotide molecule expressible in a given host comprising the sequence of the araB promoter operably linked to a gene which is heterologous to said host. The heterologous gene codes for a peptide that is biologically active. The invention also relates to a genetic construct which comprises a first genetic sequence coding for cecropin operably linked to a second genetic sequence coding for a polypeptide which is capable of suppressing the bactericidal effect of the resulting fusion protein towards an otherwise cecropin sensitive bacterium.

RELATED APPLICATION

This application is a division of application Ser. No. 07/474,304 nowU.S. Pat. No. 5,028,530, filed Feb. 5, 1990, which is a continuation inpart of application Ser. No. 07/645,309, filed Jan. 25, 1985, which isnow abandoned.

BACKGROUND OF THE INVENTION

1. Field of The Invention

The present invention relates to the field of recombinant DNA technologyand to the use of araB promoters in the expression of heterologous genesin transformed hosts. This patent also relates to the design, cloningand expression of genes coding for the bactericidal peptide cecropin andanalogues thereof.

2. Brief Description of The Background Art

Genetic information, encoded in DNA molecules, is expressed by a seriesof steps involving transcription of the DNA into mRNA and the subsequenttranslation of the mRNA into polypeptides or proteins. The expression ofthe encoded information to form polypeptides is initiated at thepromoter site, a region on the DNA molecule to which RNA polymerasebinds and initiates transcription. Promoters that have been used inrecombinant DNA methods for expressing heterologous genes include thebeta-lactamase (penicillinase) and lactose (beta-galactosidase) promotersystems (Change et al., Nature, 275: 615 (1978); Itakura et al.,Science, 198: 1056 (1977); Goeddel et al., Nature, 281: 544 (1979)) andtryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res., 8:4057 (1980); EPO Application Publication No. 0036776). Other knownpromoters include the bacteriophage lambda promoters, (P_(L)) and(P_(R)), hut, colicin E₁, galactose, alkaline phosphatase, xylose A, andtac.

The araB gene and its promoter (araB) are located in the L-arabinoseoperon. The L-arabinose operon (araBAD) in Escherichia coli and inSalmonella typhimurium has been studied. Of particular interest is theL-arabinose operon in S. typhimurium; its sequence is described inHorwitz, A. et al., "DNA Sequence of the araBAD-araC Controlling Regionin Salmonella typhimurium LT2," Gene, 14: 309-319 (1981), Lin, H.-C. etal., "The araBAD operon of Samonella Typhimurium LT2, I. NucleotideSequence of araB and Primary Structure of Its Product, Ribulokinase,"Gene, 34: 111-122 (1985); II. "Nucleotide Sequence of araA and PrimaryStructure of Its Product, L-Arabinose Isomerase," Gene, 34: 123-128(1985; III. "Nucleotide Sequence of araD and Its Flanking Regions, andPrimary Structure of its Product, L-Ribulose-5-Phosphate-4-Epimerase,"Gene, 34: 129-134 (1985). The araBAD operon contains three structuralgenes which are responsible for the initial metabolism of L-arabinose.Lee, Jar-How et al., "Genetic Characterization of Salmonella typhimuriumLT2 ara Mutations," J. of Bacteriology, 158: 344-46 (1984). L-arabinoseis first converted into L-ribulose by the araA gene product, L-arabinoseisomerase. L-ribulose is then phosphorylated to L-ribulose-5-phosphateby the araB gene product, ribulokinase. The araD gene product,L-ribulose-5-phosphate 4-epimerase, catalyzes the conversion ofL-ribulose-5-phosphate to D-xylose-5-phosphate which then enters thepentose phosphate pathway. The araBAD operon is coordinately controlledby the inducer L-arabinose and the araC regulatory gene product.

Since, in one embodiment of the invention disclosed and claimed herein,the araB promoter is operably linked to the gene coding for cecropin andinserted into a suitable host for expression of the cecropin protein, itis worthwhile to review background references on cecropins.

The immune system of the Cecropia moth and several lepidopteran insectsis characterized by an effective humoral response which is mainlyassociated with the cecropins, a recently discovered family ofantibacterial peptides (Boman, H. G. and Steiner, H., Current Topics InMicrobiology And Immunology, 94/95: 75-91 (1981)). Three majorcecropins, A, B and D, have been purified from immune hemolymph andtheir sequences have been elucidated (Steiner, et al., Nature, 292:246-248 (1981); Qu, et al., European Journal of Biochemistry, 127:219-224 (1982)l; Hultmark, D., Ibid, 127: 207-217 (1982); and Hultmark,U.S. Pat. No. 4,355,104). All cecropins are small basic peptides with ahigh degree of mutual sequence homology. The amino acid sequences ofcecropins B and D from Antheraea pernyi (A.p.) and from Hylophoracecropia (H.c.) are as follows: ##STR1##

The cecropins are similar in structure to the bee venom toxin melittin,but have a broader antibacterial spectrum than mellitin, and do not lysecultured liver cells, sheep erythrocytes or insect cells. As shownabove, the carboxy terminus in all cecropins is blocked and, in the caseof cecropin A, the blocking group is a primary amide (Andreu, et al.,Proceedings of the National Academy of Sciences, USA, 80: 6475-6479(1983)). Cecropin A and several related peptides have recently beensynthesized by solid phase techniques and have been shown to be totallyindistinguishable from natural cecropin A by chemical and physicalcriteria (Andreu, et al., supra).

Interestingly, the carboxy terminal tetrapeptide imide was found to beof little importance for the antibacterial activity towards E. coli, butfor three other bacteria tested, the activity was reduced to 3% to 20%of that of cecropin A.

The cecropins are antibacterial against a variety of bacteria includingboth Gram-negative and Gram-positive bacteria. The available data on themode of action of the cecropins indicate that they disrupt thecytoplasmic membranes of bacteria (Steiner, et al., Nature, 292: 246-248(1981)). It is apparent from the literature that different bacterialspecies have different sensitivities to the cecropins, and that eachcecropin has a distinct spectrum of activity. For example, Bacillusmegaterium is highly sensitive to cecropins A and B, but relativelyresistant to cecropin D. Both Gram-negative and Gram-positive organismshave been shown to be sensitive to cecropins in the micromolarconcentration range. Organisms showing a high level of sensitivity tocecropins include E. coli, Pseudomonas aeruginosa, Serratia marsescens,Xenorhabdus nematophilus, B. megatherium, and Micrococcus luteus.Although cecropins A and B show a total of twelve amino acidreplacements, their activities against nine different bacterial speciesare very similar, suggesting that many amino acid substitutions can betolerated without altering the biological activity of the peptide.Similarly, cecropin B from the Chinese oak silk moth (A. pernyi) differsfrom cecropin B from North American silk moths (H. cecropia) at fourpositions; however, three of the changes are replacements for thecorresponding amino acids found in the H. cecropia A form. The fourthchange is in a position where H. cecropia A form. The fourth change isin a position were H. cecropia A and B therefore apparent that uniquederivatives of the cecropins created by conservative amino acidsubstitutions would retain their biological activity. Non-conservativechanges such as those found in cecropin D might be expected to alter theactivity of the peptide. Cecropin D has almost as much activity againstE. coli as cecropins A and B, but has significantly reduced activityagainst eight other species of bacteria.

In view of the great usefulness of the cecropins and analogues thereofand of the great promise that recombinant DNA methods offer for theproduction of proteins, it appeared desirable to provide a system forthe production of cecropins by means of such technology.

SUMMARY OF THE INVENTION

It has now been found that the araB promoter can be used as a promoterin recombinant DNA molecules such that the araB promoter is operablylinked to a heterologous gene that codes for a biologically activeproduct. Using the araB promoter to control the expression ofheterologous genes has many attendant advantages. The araB promotercontrol system is tightly regulated. The araB promoter is inducible withL-arabinose; e.g., produced polypeptides are not synthesized prior toaddition of L-arabinose to the culture media. Thus, in the absence ofL-arabinose there is no expression of the heterologous gene to form thepolypeptides. Upon induction with L-arabinose, the polypeptide istranscribed as part of a messenger RNA which initiates at the araBpromoter. Once induced, polypeptides are produced quickly andefficiently. Furthermore, the fermentation period is brief as comparedwith other systems. Importantly, the extent of expression is increased,i.e., the level of production of the heterologous polypeptide is greatlyimproved, thus making it desirable for commercial use. Finally, theexpressed fusion polypeptides form inclusion bodies within the host cellthat remain very stable, regardless of increased size, and are amenableto purification for heterologous protein.

According to the present invention there is thus provided apolynucleotide molecule expressible in a given host comprising thesequence of the araB promoter operably linked to a gene which isheterologous to said host. The heterologous gene codes forpolypeptide(s) that is/are biologically active. The invention alsoprovides for vehicles capable of replication and expression comprisingsaid polynucleotide molecule; hosts transformed with said vehicle; andfermentation methods for cultivating said hosts for ultimate expressionand recovery of the heterologous peptide(s).

The present invention also provides a process, and tools for use of theprocess, for the production of cecropin peptides and analogues thereofby recombinant DNA methodology. The cecropins can be expressed by usingthe aforementioned araB promoter or any other promoter.

In particular, the invention provides gene sequences coding for peptideshaving cecropin-like bactericidal activity. These gene sequences, whichmay be provided alone or as part of longer sequences comprising thececropin peptide together with other peptides or amino acid residues,are, ideally, designed by computational methodology so as to optimizetheir acceptability by E. coli as expression hosts.

In particular, the invention provides a genetic construct capable ofexpression in a cecropin-sensitive host which is a fusion sequencebetween a first genetic sequence coding for cecropin operably linked toa second genetic sequence coding for a different polypeptide, whereinsaid different polypeptide is capable of suppressing the bactericidaleffect of the expressed fusion product towards said cecropin-sensitivehost.

In another embodiment, the invention provides a genetic constructcapable of expression in a cecropin-sensitive host which comprises agenetic sequence coding for cecropin operably linked to an induciblepromoter sequence.

The invention also provides vehicles capable of replication andexpression comprising the aforementioned genetic constructs, hoststransformed with said vehicles, as well as methods of producingcecropins using the aforementioned vehicles, constructs and hosts.

The invention further provides a fusion protein of a cecropin with apolypeptide wherein said fusion protein has decreased bactericidaleffects toward a given bacterial host.

Preferably, the genetic sequences coding for a cecropin are designed bycomputational methodology so as to optimize their acceptability by E.coli as expression hosts. Design optimization is also carried out tominimize self-complementarity, to avoid or create restrictionendonuclease sites within or outside the sequence and thus facilitateinsertion, and to minimize complementarity with the desired expressionvehicles. By subjecting the polynucleotide sequence to suchoptimizations, the invention provides synthetic sequences which, in mostinstances, are structurally different from those in the natural genes ofthe various species used as sources for cecropin.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid and nucleotide sequences of cecropin A andthe synthetic gene used for expression in E. coli.

FIG. 2 shows the construction of plasmid pING 1 from plasmid pMH6 andM13mp9. pING 1 carries both the Salmonella typhomurium araB and araCgenes from plasmid pMH6. The Figure also shows the construction ofplasmids pCA1A, pCA1B and pCA1' from plasmid pING1 (as the source of thearaB and araC genes).

FIG. 3 shows the construction of plasmids pCA3', PCA3A, PCA3B and PCA2'and PCA2A from pMH6 and pCA1A, pCA1B and pCA1'. The differences betweenpCA1' (or pCA1A and 1B), pCA2' (or pCA2A) and pCA3' (or pCA3A and pCA3B)lie in the varying length between the cecropin A gene and the BamHI sitebetween both ara genes, being 500 bp in pCA1' (or pCA1A and 1B), 1253 bpin pCA2' (or pCA2A) and 1550 bp in pCA3' (or pCA3A and 3B).

FIG. 4 shows the construction of plasmid p19C.

FIG. 5 shows the construction of plasmid pCA3A- -1 from pCA3A. TheFigure also shows the construction of plasmid pCA3D from pCA3A- -1 andp19C (FIG. 4).

FIG. 6 shows the amino acid and nucleotide sequences of cecropin A, asecond chemically synthesized gene used for expression in E. coli.

FIG. 7 shows the amino acid and nucleotide sequences of wild type andmutant cecropin A where

(1) denotes CA wild type (pCA3D);

(2) denotes CA mutant A (pCA3A); and

(3) denotes CA mutant B (pCA3B);

denotes the position of mutation.

FIG. 8 shows the construction of the plasmid phTGF5 containing anaraB-hTGF fusion.

FIG. 9 shows the construction of the plasmid pTGF58.

FIG. 10 shows the construction of two plasmids, p115 and p318, carryinga human synthetic calcitonin gene.

FIG. 11 shows the construction of a plasmid, pHCT1, containing the genefor the fused polypeptide ribulokinase-human calcitonin (hCT).

FIG. 12 shows the construction of plasmid pING1, used as a vehicle forcloning and expression of calcitonin-peptide genes; pING1 carries araBand araC genes.

DEFINITIONS

In the description that follows, a number of terms used in recombinantDNA technology are extensively utilized. In order to provide a clearerand consistent understanding of the specification and claims, includingthe scope to be given such terms, the following definitions areprovided.

Operon--A gene comprising structural gene(s) for polypeptide expressionand the control region which regulates that expression.

Operator--A DNA sequence capable of interacting with a specificrepressor, thereby controlling the functioning of adjacent gene(s).

Promoter--A DNA sequence within the control region at which RNApolymerase binds and initiates transcription of an adjacent gene(s).

Activator--A protein required for initiation of RNA synthesis by RNApolymerase.

Initiator--A DNA sequence with which an activator interacts to controladjacent genes.

Polynucleotide molecule--A linear sequence of nucleotides linkedtogether by a backbone consisting of an alternating series of sugar andphosphate residues and as used herein can include DNA and RNA polymers.

Structural gene--A DNA sequence which encodes through its template ormessenger RNA a sequence of amino acids characteristic of a specificpeptide.

Heterologous gene--A gene that is foreign, i.e. originating from a donordifferent from the host or a chemically synthesized gene and can includea donor of a different species from the host. The gene codes forpolypeptides ordinarily not produced by the organism susceptible totransformation by the expression vehicle.

Biologically active--As used herein means the quality or process ofaccomplishing an intended effect occurring in a biological system.

Operably linked--As used herein means that the promoter controls theinitiation of the expression the polypeptide encoded by the heterologousgene.

Expression--Expression is the process by which a structural geneproduces a polypeptide. It involves transcription of the gene intomessenger RNA (mRNA) and the translation of such mRNA intopolypeptide(s).

Cloning vehicle--A plasmid or phage DNA or other DNA sequences which areable to replicate in a host cell, which are characterized by one or asmall number of endonuclease recognition sites at which such DNAsequences may be cut in a determinable fashion without loss of anessential biological function of the DNA, and which contain a phenotypicselection marker suitable for use in the identification of transformedcells. Markers, for example, are tetracycline resistance or ampicillinresistance. The word "vector" is sometimes used for cloning vehicle.

Expression control sequence--A sequence of nucleotides that controls orregulates expression of structural genes when operably linked to thosegenes. They include the lac system, the trp system, major operator andpromoter regions of phage lambda, the control region of fd coat protein,and other sequences known to control the expression of genes inprokaryotic or eukaryotic cells.

Expression vehicle--A vehicle similar to a cloning vehicle but which iscapable of expressing a given structural gene in a host, normally undercontrol of certain regulatory sequences.

Host--Any organism that is the recipient of a replicable expressionvehicle, including bacteria and yeast.

Cecropin--This term as used throughout the specification and claims ismeant to include a polypeptide from any insect species, which hasbactericidal activity in an in vivo or in vitro system acceptable by theart to measure such activity.

The term cecropin is also used in this invention to include anyanalogue, homologue, mutant, isomer or derivative of anaturally-occurring cecropin, and which shows bactericidal activity inan appropriate system. The term is also meant to include fragmentshaving less than the naturally-occurring number of amino acids, such aspartial fragments of natural cecropins or their analogues. The term isalso used to include any product which comprises the sequence of anaturally-occurring cecropin or analogue thereof, together with one ormore flanking amino acids, preferably at the carboxy terminus, whichshow cecropin-like bactericidal activity. The term is also meant toinclude cecropins having less than the number of naturally-occurringamino acids but which still show bactericidal activity.

The degree of homology which brings a cecropin within the scope of thisdefinition will vary depending upon the cecropin regions responsible forbactericidal activity; domains which are critical for bactericidalactivity will exhibit a high degree of homology in order to fall withinthe definition, while sequences not involved in maintaining bactericidalconformation or in effecting receptor binding may show comparatively lowhomology. In addition, critical domains may exhibit bactericidalactivity and yet remain homologous as defined herein if residuescontaining functionally similar side chains are substituted."Functionally similar" refers to dominant characteristics of the sidechains such as basic, neutral or acid, or the presence or absence ofsteric bulk. Generally, a peptide defined as a cecropin will containregions substantially homologous with those of the cecropins shown inthe section of this application entitled "Description of The BackgroundArt." Less homology is required in the amino terminal region than in thecarboxy terminal region.

By "bactericidal activity" is meant to include activity as definedpreviously, which can either be greater or lesser than that ofnaturally-occurring cecropin species. In particular are includedcecropin peptides having bactericidal activity which ranges from about1% of the naturally-occurring species to activities which may besubstantially higher (e.g., 10-fold, 100-fold, or higher) than those ofthe naturally-occurring cecropins.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. The araB Promoter

The present invention provides for a polynucleotide molecule expressiblein a given host comprising the sequence of the araB promotor operablylinked to a gene which is heterologous to said host. The heterologousgene encodes for polypeptide(s) that are biologically active. Theinvention also provides for associated constructs with the araBpromoter. The araB promoter is an inducible promoter, i.e., theexpression of the heterologous gene to form the encoded polypeptide isinitiated or induced by the addition of L-arabinose. L-arabinoseinteracts with an activator, the product of the araC gene, to regulateexpression of araB. This is a positive control system in contrast to anegative control system; for example, lac, trp, lamda P_(L), and tac.Without the L-arabinose addition, the heterologous polypeptide will notbe expressed or synthesized. Once induced, the biologically activepolypeptide product(s) are produced quickly and efficiently. Thesepolypeptide product(s) typically form as inclusion bodies within thehost cells. The inclusion bodies enlarge rapidly as the cell densityincreases. The inclusion bodies remain very stable within the hostcells. This characteristic results in a reproducible high yield andrenders the fermentation monitoring an easy task. To terminate anon-going fermentation run, an accurate decision can be made simply basedon the size of inclusion bodies and saturated growth.

The araB promoter and inferred amino acid sequence for the initialportion of the araC gene in S. typhimurium and plasmids pMH6 and pING1has the following nucleotide sequence: ##STR2##

The araB promoter as part of the araB gene is located in the L-arabinoseoperon. The L-arabinose operon (araBAD) has been isolated in E. coli andS. typhimurium. However, the practice of this invention is not limitedto these two sources for the araB promoter. Other sources can includeany organism which contains genes coding for the L-arabinose operon.These sources for the araB promoter can include the genera Pseudomonas,Citrobacter, Xanthomonas, and Erwinia.

In addition, the araB promoter may be synthesized; e.g., by manipulationin the laboratory, rather than of natural origin. In other words, theconcept connotes an artificially compounded or even an artificiallydegraded product. Thus, even though a given complete sequence for anaturally-occurring araB promoter may exist integrated into the genomicDNA of a given organism species, the isolated sequence corresponding tothe araB promoter separated from the genomic DNA of the organism, withor without adjacent sequences corresponding to leading polypeptides,start or stop signals, is not naturally occurring and would beconsidered to be "synthetic." In addition, given the degeneracy of thegenetic code, the knowledge of an amino acid sequence does notnecessarily and irrevocably lead to the naturally occurring geneticsequence coding therefor.

Optimization of any synthetic sequence comprises four or more possiblesteps and is applicable to the synthesis of the araB promoter, as wellas to synthesis of peptide sequences, including heterologouspolypeptides. First, for any given desired sequence, a list of possibleDNA codons for each amino acid in such sequence is generated, with thosecodons ranked in order according to the frequency with which they areused in bacteria or yeast. A preliminary ordering of the codons might bebased on the paper by Bennetzen and Hall, Journal of BiologicalChemistry, 257: 3026- 3031 (1982) (yeast) or Fiers, W., Nature, 260: 500(1976) (bacteria), herein incorporated by reference. A furtherrefinement of the sequence beyond the techniques of these papers is alsorecommended.

A second factor which influences the choice of any particular codon isthe presence or absence of certain restriction enzyme sites that mightbe used in the process of clonging the gene, so that the use of thoseenzymes during the cloning process would be facilitated. Optimization ofthis factor comprises comparing the known endonuclease site sequences(comprising four to six nucleotides per site) with the primary"host-preferred" sequence. A list of restriction endonuclease sites canbe found, for example, in Roberts, R. J., Nucleic Acid Research, 11: 1(1983), r135-r137.

A third factor which influences the choice of particular codons is theneed to minimize the internal secondary structure of the synthesized DNAfragments, to prevent them from folding upon themselves and inhibitingthe annealing reactions to adjacent DNA fragments. This factor comprisessearching a given design sequence so as to avoid undue complementarityof segments thereof, one with another, save for segments adjacent to oneanother in the intended gene. A search for complementarity (i.e.,avoidance thereof) can also be carried out between the designed genesequence and proposed replication vehicles, such as plasmids or phages.

A fourth factor involved in the optimization is the avoidance ofsequences rich in AT base pairs (about 5 or more), especially whenpreceded by a sequence rich in GC base pairs, to avoid prematuretermination of transcription.

A fifth factor influencing the choice of codons is the avoidance ofRNase sites so that the message is stable.

Finally, if any of two possible codons could be used, it is preferred toutilize that which will maximize expression in microbial genomes (see,for example, Fiers, et al., Nature, 260: 500 (1976); Grosjean, et al.,Gene, 18: 199-209 (1982); and Riggs U.S. Pat. No. 4,366,246, column 6,all of which are herein incorporated by reference).

Most preferably, a computer program designed to carry out the necessarycomparisons, to optimize expression in bacterial microbes or yeast, isutilized for the optimization.

In one embodiment of this invention, the inducible araB promoter isoperably linked to a genetic sequence coding for a polypeptide that isbiologically active, and the resulting genetic construct is introducedinto or forms part of an expression vehicle. The expression vehicle isthen utilized to transform an appropriate host. The host is fermentedunder selected culturing conditions to achieve optimum growth. The araBpromoter is not active until treated with L-arabinose, which induces thepromoter to initiate expression of the heterologous gene. The sequenceof actions include transcription of the gene into mRNA, and thetranslation thereof into the polypeptide product(s).

In another embodiment of this invention, the araB promoter is operablylinked to a genetic sequence coding for a heterologous polypeptide thatis biologically active, and this genetic sequence is operably linked toa second genetic sequence coding for another polypeptide. The expressionyields a fusion or precursor protein comprising both the amino acidsequence of the second polypeptide and that of the desired heterologouspolypeptide, and containing a selective cleavage site adjacent to thedesired amino acid sequence.

The cleavage site is preferably methionine, although the site may be anypreferred site known in the art. The desired heterologous polypeptideshould preferably lack internal cleavage sites corresponding to theactual selected cleavage site. Other known cleavage sites includeAsn-Gly, Asp-Pro, Lys, Arg, and Lys-Arg.

Selective cleavage of the fusion or precursor protein is typicallyeffected outside of the replicative environment of the expressionvehicle. In this posttranslational step, the fusion or precursor proteinis clipped by selective treatment. For example, when methionine is thecleavage site, the fusion or precursor protein is treated with cyanogenbromide to clip the desired heterologous polypeptide. With other knowncleavage sites, the clipping treatment includes hydroxylamine, acid,trypsin, and Lys-Arg cleavage enzyme.

Methods for preparing fused, operably linked genes and expressing thesame in bacteria are known and are shown, for example, in U.S. Pat. No.4,366,246, herein incorporated by reference.

The genetic constructs and the methods involved herein can be utilizedfor expression of the heterologous polypeptides in bacterial hosts.

For example, E. coli K12 strain 294 (ATCC 31446) and strain MC1061 (ATCC39450) are particularly useful. Other microbial strains which may beused include, but are not limited to, E. coli X1776 (ATCC 31537). Theaforementioned strains, as well as E. coli W3110 (F⁻, lambda⁻,prototrophic (ATCC 27325)), and other enterobacteriaceae such as S.typhimurium or Serratia marcescens, and various pseudomonad species maybe used.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as specific genes which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (Bolivar, et al., Gene, 2: 95 (1977)). pBR322 contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells. The pBR322 plasmid, or other microbialplasmids must also contain, or be modified to contain, promoters whichcan be used by the microbial organism for expression of its ownproteins.

The araB promoter may be operably linked to a genetic sequence codingfor any heterologous polypeptide or protein that is biologically active.Examples of such polypeptides or proteins include, but are not limitedto, enzymes, hormones, hemoglobin, antibodies, structural proteins,alpha, beta and gamma interferons, interleukins, insulin, and tissueplasminogen activators. Specific examples include human tumor growthfactor, calcitonin, and cecropin.

The transformed host can be fermented and cultured according to meansknown in the art to achieve optimal cell growth. The preferredfermentation and production procedure of this invention, describedbelow, can be used to achieve large scale production of heterologouspolypeptides. In addition, by using the araB promoter operably linked tothe heterologous gene, the extent of expression of the heterologous geneis increased.

The preferred fermentation procedure is as follows: The transformedhost, preferably bacteria, more preferably E. coli, is introduced into aculture medium containing nutrient materials that meet the growthrequirements of the bacterium. Such materials may include carbon andnitrogen sources, minerals, amino acids, purines, pyrimidines, andvitamins. The preferred culture medium also comprises a metabolite in anamount sufficient for phenotypic marker resistance, such metabolitebeing, for example, tetracycline or ampicillin. The host is grown underculturing conditions selected to achieve maximum growth rate.Temperature conditions will depend upon the host, but typically theoptimum range is about 30° C. to about 40° C., with 37° C. being themost preferred for transformed E. coli. Oxygen is also provided to themedium. The host is allowed to grow until late exponential phase andthen transferred to fresh culture medium.

The hosts are then inoculated with production culture medium. Duringthis step, the bacteria will continue to divide and grow until thebacteria reach a concentration, saturation density, at which thebacteria no longer divide, but are still viable. The time sufficient toreach the saturation density is dependent on the medium, the genotype ofthe host, the temperature, and the degree of aeration.

Initially, the fermentation and culturing conditions for the productionstep are the same as those given above for the culture step, except thatas dissolved oxygen is consumed during growth, the agitation andaeration rate are increased accordingly to maintain a minimum dissolvedoxygen (D.O.) at 30%. The preferred pH range is between about 6 to 8.The pH of the above-described production medium is consistentlyself-adjusted at 6.5-6.8, which is optimum for E. coli growth. Thus,acid and base control of the pH medium is unnecessary for the E. colihost; however, it may be necessary to adjust the pH medium for otherhosts. When optimal cell density is reached, optimally OD₆₀₀ ¹⁰ for E.coli, L-arabinose is added in an amount sufficient to induce thesynthesis of the heterologous gene to form the polypeptide. As describedabove, the polypeptide forms inclusion bodies within the host. Thepeptide is then recovered according to means known in the art such asfiltering or precipitation. If a fusion protein is the resultingexpressed heterologous peptide, the fusion protein is recovered and thenmay be treated in a post-translation step to separate the desiredheterologous polypeptide. The heterologous polypeptide can then berecovered and purified according to known means.

The use of the araB promoter will be described, for convenience,relating to various embodiments of the invention comprising cecropin,although it is to be understood that the araB promoter may be operablylinked to a genetic sequence coding for any polypeptide or protein thatis biologically active.

2. Methods of Microbial Production of Cecropins Using Cecropin-SensitiveHosts

Part of the present invention also provides methods of microbiologicalproduction of cecropins using cecropin-sensitive hosts. One concept ofthe invention is to express a gene sequence coding for cecropin while,simultaneously, avoiding or delaying the bactericidal effects of theproduct.

In a one embodiment, the genetic sequence coding for cecropin is linkedto an inducible promoter, and the resulting genetic construct isintroduced into an expression vehicle. The expression vehicle is thenutilized to transform an appropriate host and the host is fermentedunder normal conditions wherein the promoter is not active. After anappropriate period of time, such as, for example, at a time when thecells are in stationary phase, the promoter is induced as by varying anoutside environmental factor such as salt concentration, light, presenceor absence of a metabolite, a metal, and the like, this change leadingto transcription of the cecropin genetic sequence into mRNA, and thentranslation thereof into bactericidal cecropin. Even though theresulting cecropin is bactericidal and destroys the bacterial host, suchdestruction does not occur until late in the fermentation cycle.Examples of regulated promoters are lambda P_(L) and P_(R), lac, gal,trp, ara, hut, and the like.

In the second preferred embodiment, it has been discovered that if agenetic sequence coding for cecropin is operably linked to a polypeptideother than said cecropin, such that the expression yields a fusion orprecursor protein comprising both the amino acid sequence of cecropinand that of the additional polypeptide, and containing a selectivecleavage site adjacent to the desired cecropin amino acid sequence, theresulting fusion protein is not bactericidal. Bactericidally activececropin can then be isolated post-translation by selective cleavage.

Most commonly, cleavage will be effected outside the replicativeenvironment of the expression vehicle, such as, for example, followingharvest of the microbial culture. Thus, the additional polypeptide robsthe cecropin of its bactericidal effect pending extracellular cleavage,allowing the survival of the host for long enough time to yield highlevels of the desired product. Preferably, the cecropin will lackinternal cleavage sites corresponding to the selective cleavage siteemployed to shed the superfluous polypeptide, although this is notnecessarily an absolute condition. Since the cecropins are methioninefree, cyanogen bromide cleavage at the methionine adjacent to thececropin sequence is effective.

Preferably, the genetic sequence coding for the superfluous polypeptideis transcribed in advance of the structural gene of the cecropin, butthis need not necessarily be the case, as it may also be possible toexpress the superfluous polypeptide in a position adjacent to theC-terminal of the cecropin.

The nature of the superfluous adjacent polypeptide is not critical. Itcould either be part, whole or repetitive units of a known structural,enzymatic, hormonal, or other physiologically relevant proteins.Alternatively, it could be a non-functional polypeptide. Without beingbound by any particular theory, the inventors speculate that theincreased length of the fusion protein somehow "masks" the bactericidalproperties of the cecropin due to the varying conformation of theoverall polypeptide. Preferably, the genetic sequence coding for theadjacent superfluous polypeptide should be at least about greater than300 base pairs in length, preferably between about 400 bp and 5 Kb, mostpreferably between 400 bp and 2 Kb. This corresponds to a superfluouspolypeptide of preferably between about 100-1700 amino acid residues.

Any of a large number of superfluous polypeptides can be fused to thedesired cecropin peptide sequence. The polypeptide gene sequence caneither be prepared by organic synthesis, in which case optimizationprocedures would be recommended, or might be prepared by such techniquesas reverse transcription of appropriate mRNA. Enzymatic coupling of thegene sequence for the polypeptide to the gene sequence for thestructural cecropin peptide would then follow the preparation of thecDNA. Enzymatic coupling can be either by blunt ligation or by theprovision of cohesive termini, comprising one of the two strands of arestriction endonuclease recognition site. Examples of superfluouspolypeptides are beta-galactosidase or ribulokinase (encoded for by thearaB gene). Enzymes and structural proteins are preferred. Otherproducts which can be used are products encoded by the following genes:aceA or aceB, araA, araB, araC, araD, argG, aroB, lacA, serA, purA,trpA, trpB, trpC, trpD, trpE, tyrA, and the like. The superfluouspolypeptide is normally free of glycosylation. The araB promoter is thepreferred promoter, although other promoters such as lambda P_(L) andP_(R), lac, gal, trp, hut, and other ara promoters may be used.

In yet another embodiment of the invention, the cecropin geneticsequence is operably linked to the sequence for a superfluouspolypeptide capable, in the fusion product, of inhibiting orinactivating the bactericidal activity of cecropin and, in addition, toan inducible promoter. In this manner, both the effect obtainablethrough the fusion protein technique and the effect obtainable throughthe use of the inducible promoter can be exploited advantageously andsimultaneously.

Although the present invention results in methods for producingcecropins in cecropin-sensitive hosts, e.g., bacterial hosts, thegenetic constructs prepared herein and the methods involved can also beutilized for expression of cecropin in other, non-cecropin sensitivehosts. These non-cecropin sensitive hosts include yeasts and mammaliancell cultures. Useful yeast and mammalian hosts and vectors are wellknown to those of skill in the art, and reference is made, for example,to European Patent Publication 0093619 published Nov. 9, 1983. Bacterialhosts can include those mentioned hereinabove with the araB promoter, aswell as bacterial hosts such as the genera Pseudomonas, Citrobacter,Xanthomonas, and Erwinia. Any plasmid vector compatible with thesehosts, as described above with the araB promoter, can be used.

Another preferred promoter for cecropin is lambda (P_(L)). The geneticconstruct for cecropin and the superfluous polypeptide can be placedunder the control of the leftward promoter of bacteriophage lambda(P_(L)). This promoter is one of the strongest known promoters which canbe controlled. Control is exerted by the lambda repressor, and adjacentrestriction sites are known. A temperature sensitive allele of CI genecan be placed on the vector that contains the cecropin complete sequenceor a different vector. When the temperature is raised to 42° C., therepressor is inactivated, and the promoter will be expressed at itsmaximum level. The amount of mRNA produced under these conditions shouldbe sufficient to result in a cell which contains about 10% of its newlysynthesized RNA originated from the P_(L) promoter. In this scheme, itis possible to establish a bank of clones in which a functional cecropinfusion construct sequence is placed adjacent to a ribosome bindingsequence, and at varying distances from the lambda P_(L) promoter. Theseclones can then be screened and the one giving the highest yieldselected.

The expression of a cecropin sequence can also be placed under controlof other regulons which may be "homologous" to the organism in itsuntransformed state. For example, E. coli chromosomal DNA contains alactose or lac operon which mediates lactose digestion by elaboratingthe enzyme beta-galactosidase. The lac control elements may be obtainedfrom bacteriophage lambda plac5, which is infective for E. coli. Thephage's lac operon can be derived by transduction from the samebacterial species. Regulons suitable for use in the process of theinvention can be derived from plasmid DNA native to the organism. Thelac promoter-operator system can be induced by IPTG.

Of particular interest in the present invention is to provide syntheticpolynucleotide sequences coding for the cecropin peptides. In apreferred embodiment of the present invention, the synthetic sequence ofthe cecropin peptide (with or without adjacent sequences) is optimizedso that expression thereof will be compatible with a variety of hostssuch as yeasts and bacteria, especially the latter. In particular,optimization of the expression of any given sequence in E. coli is ofgreat importance. Thus, after performing such optimization procedures asindicated above, the actual genetic sequence of the cecropin peptideswith or without adjacent sequences, will usually be distinct from thenaturally-occurring sequence in the original species.

In addition, the design of the desired gene for the fused product shouldpreferably incorporate codons for amino acids at the cleavage site asmethionine (cleavable by cyanogen bromide), tryptophan (cleavable byo-iodosobenzoic acid), glutamic acid (cleavable by Staph. protease) andthe like.

In one embodiment of the invention, any given codon in the desired DNAsequence for the fusion product can be mutagenized at will throughsitedirected mutagenesis. Thus, it is possible, after synthesis of thedesired DNA sequence, to introduce into the sequence a cleavable site.Site-directed mutagenesis is known, and reference is made to Wallace etal., Science, 209: 1396-1400 (1980), herein incorporated by reference.

The amino acid residue or residues following the potential C-terminalresidue in the cecropin-peptide may be followed by yet anotherpolypeptide ("trailing" polypeptide) of varying length and of structuresimilar or different than a leading polypeptide if one is present, asdescribed above. In such case, it is convenient to provide for the sameor different selective cleavage sites between the C-terminal amino acidresidue and the trailing polypeptide sequence. These cleavable sites mayor may not be the same as those present between the leading polypeptideand the structural gene for the cecropin peptide.

In other words, the superfluous polypeptide may be present as a leadingpeptide, a trailing polypeptide or both.

Where the structural gene of the desired cecropin peptide is to beinserted into a vehicle for expression as such, the gene would bepreceded by a "start" codon, and if followed by trailing sequences, oneor more termination or stop codons. When the expression product is afusion protein comprising both the cecropin peptide and part or whole ofa polypeptide, the start codon may be placed prior to the N-terminus ofthe polypeptide if it is leading.

Methods for the syntheses of polynucleotides are well known to oneskilled in the art. Reference is made, for example, to the triestermethod of Itakura et al., Journal of American Chemical Society, 97: 3727(1975).

The cecropins produced by the methods of the invention can be used asbroad anti-microbial agents directed toward specific applications. Suchapplications include, for example, the use of the cecropins aspreservatives in processed meat products (target organisms: (1)Clostridium botulinum, (2) Lactobacilli, (3) Micrococci); anti-cariesagents in oral hygiene products (target organism: Streptococcus mutans);agents useful in the treatment of vaginal yeast infections (targetorganism: Candida albicans); and as anti-bacterial agents in deodorants(target organisms: (1) Micrococci, (2) Diphtheroids). The relativeeffectiveness of cecropin-like peptides for the applications describedcan be readily evaluated by one of skill in the art by determiningsensitivity of any organism to one of the cecropin peptides. The samebacterial screen used in vitro can be utilized to determine dosages andconcentrations.

Having now generally described this invention, the same will be betterunderstood by reference to a specific example which is included hereinfor purposes of illustration only and is not intended to be limitingunless otherwise specified.

METHODS Amino Acid Compositions

Hydrolysis of cecropin polypeptides was accomplished using 5.5M HCl(Pierce) at 110° C. for 24 hours in vacuo. Samples were dried undervacuum at 40° C. and resuspended in 200 ul of Beckman Model 6300 samplebuffer (low pH citrate).

Amino acid analysis was performed using a Beckman Model 6300 analyser.Postcolumn ninhydrin was used for detection. Data was recorded using aHewlet Packard (HP) integrator Model 3390. The individual peaks wererecorded as the sum of the signals at 540 and 440 nm. Beckman standardscontaining each amino acid were used to calibrate the integrator andsperm whale myoglobin (Applied Biosystems) was used to verify thecalibration.

Protein Sequencing

An Applied Biosystems Model 470A protein sequenator was used for allprotein sequence determinations. Specifications for the sequencingmethodology follow the Hunkapiller-Hood program. The system uses anon-vacuum program with cleavage by methanolic-HCl.

PTH Determinations

PTH determinations were accomplished using an HP model 1090A HPLC withan HP model 3390A integrator for data presentation. An IBM cyano-propylcolumn was used for separation of the PTH amino-acids. The buffer was 30mM NaHAc pH 5.1 containing 5% tetrahydrofuran. The flow rate was 1ml./min. and temp. 37° C.

Samples were dissolved after drying in vacuo with 30 ul of 33%acetonitrile in water for 30 min. The PTH standards used were fromPierce Chemical Co.

EXAMPLE 1 Synthesis and Cloning of a Synthetic Cecropin A (CA) Gene

A. Synthesis of the CA Gene

A gene coding for CA was designed by computer to incorporate codonsnormally found in highly expressed E. coli proteins. At the end of thegene were incorporated 4-bp overhangs to permit the ligation of SalI andEcoRI sites respectively. To facilitate the construction of varioussizes of fused proteins a BamHI site was included following the SalIsite. Also, by using a computer program, optimization procedures asdescribed previously were taken into account. The gene was divided intoeight oligonucleotides ranging from 23 to 37 bases in length (FIG. 1).

The eight single-stranded DNA fragments were synthesized according tothe solid-phase phosphotriester method as described by Ito, et al.,Nucleic Acids Research, 8:5491 (1982).

Several modifications and improvements were made, including thefollowing:

(1) The coupling reaction was carried out at 37° C. for 40 minutes withgentle shaking.

(2) For the deprotection of the DNA after final coupling reaction, theDNA resin (10 mg) was reacted with a 0.5M 2-pyridinealdoxime-tetramethylguanidinium solution (2 ml) in pyridine-water (8:2 v/v) for 60 hours at37° C. with shaking. The combined solution was evaporated and treatedwith NH₄ OH (28%, 3 ml) in a sealed ampoule at 60° C. for 36 hours.After evaporation of the ammonia, the solution was extracted with etherthree times and then evaporated. For the purification of the DNA, theresidue was dissolved in 0.1 ml of 50 mM triethylammonium bicarbonate pH7.5 (TEAB) and applied to a Sephadex G-50 column (1×50 cm). One mlfractions were collected. The first few fractions, at the leading edgeof the major 260 nm-absorbing peak, contained the desired product. Thesefractions were evaporated, then purified further by high pressure liquidchromatography (HPLC). HPLC purification was carried out on a BondapakC18^(R) (Waters) column at 55° C. using a linear gradient ofacetonitrile (10-40%) in 10 mM triethylammonium acetate buffer (pH 7.8).The DMT group was hydrolyzed by treatment with 80% acetic acid (0.1 ml)for 25 minutes at room temperature followed by evaporation and thenevaporated with 0.1 ml water to remove the acetic acid completely.

B. CA Gene Assembly by Ligation

The 5'OH termini of the chemically synthesized fragments 1 through 8were separately phosphorylated in the presence of 10 ul of solutioncontaining:

70 mM Tris-HCl (pH 7.6)

10 mM MgCl₂

5 mM dithiothreitol

66 uM gamma-³² P-ATP (1 uCi)

400 ng of DNA and 2 units of T4 polynucleotide kinase.

The reaction was held at 25° C. for 15 minutes then 1 ml of 10 mMunlabelled ATP was added to continue the reaction for another 15minutes. To check the purity, 10% of the phosphorylated DNA was analyzedby the standard method using polyacrylamide gel (15%) electrophoresis inthe presence of 7M urea.

Four hundred ng of the phosphorylated DNA fragments 2, 3, 4, 5, 6 and 7were heated at 95° C. for five minutes to inactivate the T4polynucleotide kinase. Then 400 ng of unphosphorylated fragments 1 and 8were added and heated at 95° C. for another five minutes and then cooledto 25° C. slowly (1° C. per 10 min.). The eight DNA fragments wereligated in a total volume of 100 ul in the presence of 66 mM Tris-HCl(pH 7.5), 6.6 mM MgCl₂, 10 mM dithiothreitol, 0.4 mM ATP and 2 units ofT4 DNA ligase for two hours at 25° C.

C. Construction of Plasmids pING 1, pCAlA, pCA1B and pCA1'

The construction scheme is shown in FIG. 2. The sequence of the araBpromoter is described in Horwitz, A. H. et al., Gene, 14:309-319 (1981).pMH6 is described in line, H.-C. et al., Gene 34:111-122 (1985) andM13mp9 is commercially available.

1. The CA gene fragments obtained from Step B, supra, were ligated toplasmid pING 1 which had been pretreated with restriction endonucleaseSalI and EcoRI, then digested with restriction endonuclease SmaI todecrease the transformants carrying pING 1.

2. The SmaI-treated plasmid was transformed into E. coli MC1061.

3. The colonies which contained plasmids carrying the CA gene fragmentwere identified by colony hybridization using the synthetic DNA fragment7 (FIG. 1) as a probe. Three independent clones which contained the CAfragment were found in 1,000 colonies. Each of the isolated plasmids wasdigested with SalI and EcoRI to check the size of the excised fragment.The nucleotide sequence analysis of the CA insert was performed by thedideoxy chain termination procedure of Sanger et al., PNAS, 74:5463-5467 (1977) with some modification (Wallace et al., Gene, 16:21-26(1981)). Two sequences were determined and are shown in FIG. 7(2) and7(3). The plasmids containing these sequences were designated pCA1A andpCA1B (FIG. 2). Each of these sequences differed from the wild type CAsequence at the positions indicated by the arrows in FIG. 7(2) and 7(3).

Various methods can be used to generate a wild-type sequence, forexample in vivo or in vitro recombination of pCA1A and pCA1B orscreening of additional independent clones. The plasmid containing theproposed wild type sequence is designated pCA1' (FIG. 2).

In the description that follows, plasmids with primes (') denote wildtype CA sequences, whereas plasmids without primes are derived frommutants pCA1A or pCA1B.

D. Construction of plasmids pCA2', pCA2A pCA3', pCA3A and pCA3B

This is shown in FIG. 3. pCA1' (or pCA1A or pCA1B) is digested withBamHI and, after treatment with polymerase plus dTNP, followed by SstIdigestion, yields a fragment (1) which is blunt ended at one end andcarries an SstI overhang at the other. This fragment is ligated with afragment obtained from pMH6 by NarI digestion, blunt end formation andSstI treatment, to give, after T4 ligation, pCA3' (or pCA3 or pCA3B).The fragment (1) is also ligated with a fragment obtained from pMH6 byHgiAI digestion, blunt end formation and SstI treatment, to give, afterT4 ligation, pCA2' (or pCA2A).

The desired products are sought by transforming E. coli MC1061 with theligation products. Plasmids are isolated by the minilysate procedureindicated supra. Each of the isolated plasmids is digested with BamHIand PstI respectively. Plasmids from each group having the correct sizeand correct orientation of the BamHI fragment are designated pCA2' (orpCA2A) and pCA3' (or pCA3A or pCA3B).

E. Expression of araB-CA fusion protein

E. coli cells containing araB-CA fused genes in plasmids pCA1A, pCA2A,pCA3A, pCA3B and pCA3D (note: pCA3D will hereafter interchangeably beused to denote wild type CA containing plasmid pCA3') were grown at 37°C. in media containing 1.5% tryptone, 1.0% yeast extract and 0.5% NaCl(TYE) with ampicillin at 50 ug/ml. At cell densities of OD₆₀₀ =0.2,cultures were treated with L-arabinose to a final concentration of 0.5%to induce expression of araB-CA fusion protein, and were harvested whencell densities reached OD₆₀₀ =1.5 to 1.7, by centrifugation at 4000 RPMat 4° C. for 20 minutes in a Beckman JS-4.2 rotor. Cells were washedonce by resuspending in one-half original culture volume of 50 mMphosphate buffer (pH 6.6). Washed pellets of cells containing plasmidspCA1A, pCA2A, and pCA3A were extracted in one-tenth volume phosphatebuffer and 1% sodium dodecyl sulfate (SDS) and analyzed on a 10%polyacrylamide SDS-denaturing gel. By this analysis, it was shown thatE. coli cultures containing plasmid pCA3A produced more araB-CA proteinthan either pCA1A or pCA2A-transformed culture.

F. Fractionation of induced E. coli cells containing plasmids pCA3A,pCA3B and pCA3D

Washed pellets from Step E were resuspended in one-tenth original volumeof phosphate buffer, chilled to 0° C., then the cell was broken in aFrench pressure cell. The subcellular components were then centrifugedat 4000 RPM at 4° C. for 20 minutes in the rotor. Analysis of theresulting supernatant and 4000 RPM pellet on a 10% polyacrylamide SDSdenaturing gel showed that all of the araB-CA fusion protein of pCA3A,3B and 3D were found in the 4K pellet.

G. Purification of the synthetic CA

(a) Cyanogen bromide cleavage

1. The 4K pellet fraction which contained the araB-CA fused proteinobtained from the last step was mixed with 90% formic acid to give0.7-1.1 mg/ml protein in 70% formic acid. A 10-fold excess of cyanogenbromide (1 gm/ml stock in acetonitrile) by weight was added. Thereaction mixture was flushed with nitrogen and incubated at roomtemperature overnight.

2. The formic acid and cyanogen bromide were removed under a stream ofnitrogen. The residue was dissolved in 70% formic acid and dried under astream of nitrogen twice more.

3. A solution of 0.1% trifluoroacetic acid in water was added; thisdissolved the cleaved CA completely at 1 mg protein/ml.

(b) High pressure liquid chromatography of cyanogen bromide fragments

The cyanogen bromide-cleaved fused protein was chromatographed by HPLCto isolate the fragment from the CA portion of the molecule. A C-18reverse-phase column (Waters) was used. A gradient was run with 0.1%trifluoroacetic acid (TFA) in water (buffer A) and 0.1% TFA inacetonitrile (buffer B). The starting eluent contained 20% buffer B; at2 minutes after sample injection, a gradient of 20% B to 60% B over 60minutes was initiated. The flow rate was 1 ml/minute. The elutionprofile was monitored at 280 nm. Various peaks in the chromatogram ofthe cyanogen bromide-cleaved fused protein which eluted were collectedto test the bactericidal activity as described later below (Example 4).

H. pCA3A Codes for a Mutant Cecropin Polypeptide

                  TABLE I                                                         ______________________________________                                        Amino Acid Composition of Cecropin-A                                          Produced by pCA3A in E. coli                                                               Mutant A                                                                        Experimental                                                                             Theoretical                                         Amino Acid     Values     Values*                                             ______________________________________                                        Asp            2.9        3                                                   Thr            0.1        0                                                   Glu            2.7        2                                                   Pro            2.4        2                                                   Gly            6.3        4                                                   Ala            3.0        3                                                   Val            3.6        4                                                   Ile            2.9        4                                                   Leu            1.2        1                                                   Phe            1.1        1                                                   Lys            6.2        6                                                   Arg            2.1        2                                                   Trp            ND         1                                                   Ser            1.3        1                                                   Cys            0          0                                                   Met            0          0                                                   Tyr            0.1        0                                                   His            0.4        0                                                   ______________________________________                                         N.D. = not determined.                                                        *Theoretical Values: Obtained from DNA sequencing analysis of each mutant     clone.                                                                   

The nucleic acid and the amino acid sequences are shown in FIG. 7.

EXAMPLE 2 Demonstration of Another Mutant Cecropin Polypeptide

The cells of plasmid pCA3B were fractionated, CA sequences weredetected, and the synthetic CA was purified and analyzed as shown inExample 1, Parts F-G. Amino acid composition and sequence data, shown inTable II, indicated that the second clone also contained a CA genesequence coding for a mutant cecropin.

                  TABLE II                                                        ______________________________________                                        Amino Acid Composition of Cecropin-A                                          Produced by pCA3B in E. coli                                                               Mutant B                                                                        Experimental                                                                             Theoretical                                         Amino Acid     Values     Values*                                             ______________________________________                                        Asp            2.3        2                                                   Thr            1.0        1                                                   Glu            4.2        4                                                   Pro            1.0        1                                                   Gly            4.1        3                                                   Ala            5.0        5                                                   Val            3.6        4                                                   Ile            3.9        5                                                   Leu            1.1        1                                                   Phe            0.9        1                                                   Lys            6.6        7                                                   Arg            1.0        1                                                   Trp            ND         1                                                   Ser            1.1        1                                                   Cys            0          0                                                   Met            0          0                                                   Tyr            0          0                                                   His            0.1        0                                                   ______________________________________                                         N.D. = not determined.                                                        *Theoretical Values: Obtained from DNA sequencing analysis of each mutant     clones.                                                                  

The nucleic acid and amino acid sequences are shown in FIG. 7.

EXAMPLE 3 Construction of Plasmid pCA3D Coding for Wild Type Cecropin A

The construction scheme is shown in FIGS. 4 and 5.

A. Construction of pl9C

1. Plasmid pING 1 was digested to completion with endonucleases Fokl andthen BamHl; the resulting digestion was halted by phenol/chloroform(ratio 1:1) extraction and the aqueous residue was washed with ether andthen passed through a Bio-Gel^(R) P-10 column to remove thephenol/chloroform.

The result of Fokl digestion produced a "CTAC" 5' overhang. This 76 basepair BamHI, Fokl fragment contains the araB promoter.

2. A new CA gene fragment (see FIG. 6) was constructed using the sameprocedure as described in Example 1, Step B, except that the two DNAfragments No. 1 and 5 were replaced with No. nn-1 and nn-5 (sequenceshown in FIG. 6) to produce a "GATG" 5' overhang at its N-terminus andan EcoRI site overhanging ATTT at its C-terminus.

3. pBR322 was digested to completion with the endonucleases BamHI andEcoRI. The resulting digestion was halted as described.

4. The BamHI-Fokl fragment from Step 1, supra, and the new CA gene fromStep 2, supra, were ligated to the larger BamHI-EcoRI fragment ofpBR322.

5. The ligated product was digested with endonuclease HindIII todecrease the number of transformants carrying pBR322.

6. The HindIII-treated plasmid was transformed into E. coli MC1061.

7. The colonies which contained plasmids carrying the CA gene fragmentwere identified by colony hybridization using the synthetic DNA fragment7 (FIG. 6) as probe. Three independent clones which contained the CAfragment were found in 1,000 colonies. Each of the isolated plasmids wasdigested with BamHI and EcoRI; a plasmid able to release the correctfragment as designated pl9C. The nucleotide sequence analysis of the CAinserts was performed by the dideoxy chain termination procedure ofSanger et al., PNAS, 74: 5463-6467 (1977), with some modification(Wallace et al., Gene, 16:21-26 (1981)). pl9C contained the correctsequence for the CA gene. The CA gene was placed directly downstream ofthe araB promoter without any araB coding sequence in between.

B. Construction of pCA0

1. The BamHI-EcoRI fragments of pl9C were excised by digestion withexcess amounts of restriction endonuclease EcoRI and BamHI. The plasmidswere also digested with PvuI to decrease the chance that the excisedfragments would be religated to the plasmid in the next ligation step.

2. The BamHI-EcoRI fragments from Step A.7 were ligated to plasmid pING1 which had been pretreated with restriction endonuclease BamHI andEcoRI, then digested with restriction endonuclease SmaI to decrease thetransformant carrying pING 1.

3. The SmaI-treated plasmid was transformed into E. coli MC1061.

4. The colonies which contain plasmids carrying the CA gene fragmentwere identified by plasmid characterization. Each of the isolatedplasmids was digested with BamHI and EcoRI to check the size of theexcised fragment. One plasmid that had the correct size was designatedpCA0. The nucleotide sequence analysis of the CA inserts was performedby the dideoxy chain termination procedure. This pCA0 contained thecorrect sequence for the CA gene. This pCA0 containing the complete araBregulatory gene and CA gene is placed directly after the araB promoterto express the cecropin-A directly, without creating an araB-CA fusionprotein.

C. Construction of Plasmid pCA3A-Δ-1

The purpose of this construction is to delete the AvaI-PvuII region frompCA3A in order to eliminate one Xmnl site. The deletion is also able toincrease the plasmid copy number in E. coli cells.

1. pCA3A was digested with endonuclease AvaI and PvuII and then filledin with the Klenow fragment of DNA polymerase I in the presence of dNTPto produce a blunt end.

2. The plasmid from Step 1. was religated and then digested with AvaIand PvuII to decrease the number of transformants containing originalpCA3A.

3. The AvaI, PvuII-treated plasmid was transformed into E. coli MC1061.

4. Colonies which contained plasmids deleted for the AvaI-PvuII regionwere identified by colony hydridization using the synthetic DNA fragment5'-TCATCAGCGTGGTCG-3' as a probe. Forty-six independent clones with theAvaI-PvuII region deleted were found in fifty colonies. Each of theisolated plasmids was digested with Xmnl to check the size of theexcised fragments. One of the plasmids that had the correct size wasdesignated pCA3A-Δ-1.

D. Final Assembly of Plasmid pCA3D

pl9C contains the wild-type cecropin-A sequence but lacks a convenientrestriction site at its N-terminus to create a protein fusion with araB.

pCA3A has a deletion of two base pairs occurring near the C-terminus ofthe CA gene, causing it to produce a mutant araB-CA fused protein.

These two plasmids were recombined to construct a plasmid that had thewild-type CA gene fused to araB and produce an araB-CA fused protein.

The construction scheme is shown in FIG. 5.

1. Plasmid pl9C (1 ug) was digested to completion with the endonucleaseXmnl; the resulting digestion was halted by heating at 65° C. for 10min.

2. Plasmid pCA3A-Δ-1 (0.1 ug) was digested to completion with theendonuclease Xmnl, the resulting digestion halted by heating at 65° C.for 10 min.

3. DNA from steps D.1 and D.2 were mixed and ligated.

4. The ligated product was digested with PvuII to decrease thetransformants carrying pl9C.

5. The PvuII-treated plasmid was transformed into E. coli MC1061.

6. The colonies which contained plasmids carrying the wild-type araB-CAgene and able to produce an araB-CA fused protein were identified by DNAsequence analysis, as previously described. Among seven coloniesanalyzed, one containing the wild-type sequence was obtained, anddesignated pCA3D.

                  TABLE III                                                       ______________________________________                                        Amino Acid Composition of                                                     Wild-Type Cecropin-A Produced by pCA3D in E. coli                                            Experimental                                                                             Expected                                            Amino Acid     Values     Values                                              ______________________________________                                        Asp            2.2        2                                                   Thr            1.0        1                                                   Glu            4.1        4                                                   Pro            1.1        1                                                   Gly            4.4        4                                                   Ala            5.2        5                                                   Val            3.4        4                                                   Ile            4.3        5                                                   Leu            0.7        1                                                   Phe            1.1        1                                                   Lys            7.3        7                                                   Arg            1.0        1                                                   Trp            ND         1                                                   Ser            0.3        0                                                   Cys            0          0                                                   Met            0          0                                                   Tyr            0          0                                                   His            0          0                                                   ______________________________________                                         N.D. = not determined.                                                   

The nucleic acid and amino acid sequence are shown in FIG. 7.

EXAMPLE 4 Assay of Bactericidal Activity

Agar plates (8 cm in diameter) were prepared with 6 ml of TYE mediumcontaining about 10⁷ viable cells of test organism. Wells, 2 mmdiameter, were punched in the plates. The test material was dissolved in50 mM phosphate buffer (pH 6.6) and 3 ul was applied to each well. Thediameters of the inhibition zones, or halos, around the wells weremeasured after overnight incubation at 25° C. To determine a standard ofhalo formation, CA 1-33 protein was used. 1 ug and 3 ug of CA 1-33 inwell applications caused halos with diameters of 8 mm and 11 mmrespectively, on media infused with E. coli strain JF568. 10 ug and 30ug of cyanogen bromide-digested pCA3A araB-CA fusion protein causedhalos with diameters 7 mm and 18 mm respectively.

Cyanogen bromide-digested fusion protein fractionated by HPLC was alsoapplied to this assay. Various peaks were tested, and one specific peakwas shown to be bioactive. Other bacterial and yeast strains were alsotested in this assay. Cyanogen bromide-digested and undigested fusionprotein samples were applied in the amounts described above in thetesting of E. coli strain JF568, and the resulting halo diameters weremeasured.

Results are shown for all cecropins obtained in Table IV.

                  TABLE IV                                                        ______________________________________                                        Bioactivity of CNBr-digested pCA3A, pCA3B, pCA3D                              fusion protein isolates (5 ug) and ampicillin (750                            ng) on various bacterial strains, as measured by                              halo activity.                                                                          Halo Activity in Millimeters                                        Bacterial Strain                                                                          pCA3A.sup.Y                                                                            pCA3B.sup.Y                                                                            pCA3D* Ampicillin                               ______________________________________                                        Staphylococcus                                                                            NE       NE       NE     11.5                                     aureus                                                                        Streptccoccus                                                                             NE       NE       NE     7.0                                      faecalis                                                                      Salmonella derby                                                                          5.0      5.0      5.0    6.0                                      Pseudomonas PS-9                                                                          2.0       2.25    2.0    NE                                       Klebsiella  4.5      4.0      4.5    NE                                       peumoniae                                                                     Erwinia     5.0      4.5       4.75  6.5                                      carotovora EC                                                                 Streptococcus                                                                             NE       NE       NE     12.5                                     pyogenes                                                                      Xenohabdus  3.5      3.0       2.75  12.5                                     nematophillus                                                                 Serratia marcesens                                                                        1.0      1.0      1.0    NE                                       E. coli 5506                                                                              5.0      4.5      5.0    4.5                                      E. coli 5506 DR                                                                           7.0      7.0      6.5    4.5                                      ______________________________________                                         *Wild                                                                         .sup.Y Mutants                                                                NE No halo activity                                                      

EXAMPLE 5 Fermentative Production of Cecropin and of Tumor Growth Factor

This Example describes the fermentation production of alpha-TGF (tumorgrowth factor) and of cecropin under the control of araB promoter in E.coli.

The E. coli strain MC1061 contains plasmid-borne araB promoterregulating araB-alpha-TGF (pTGF58) or araB-cecropin (pCA3D) fusion gene.The cultures were grown at 37° C., 250 rpm in 100 ml of TYE culturemedium containing ampicillin (0.1 gm/l). The cultures were grown untillate exponential phase, approximately 200 Klett units; red filter. Thecultures were then transferred to a four liter baffled shake flaskcontaining 900 ml fresh TYE medium. Incubation continued for three morehours prior to inoculation into 9 liters of production medium. Theproduction medium comprises the ingredients listed in Table V:

                  TABLE V                                                         ______________________________________                                        Base Medium        Additives                                                               Level                 Level                                      Ingredients  (g/liter) Ingredients (g/liter)                                  ______________________________________                                        casein hydrolysate                                                                         30        glycerol    16                                         yeast extract                                                                              1         CaCl.sub.2 :2H.sub.2 O                                                                    0.022                                      KH.sub.2 PO.sub.4                                                                          3         MgSO.sub.4 :7H.sub.2 O                                                                    0.25                                       Na.sub.2 HPO.sub.4                                                                         6         thiamine-HCl                                                                              0.01                                       NaCl           0.5     ampicillin  0.1                                        NH.sub.4 Cl  4                                                                ______________________________________                                    

Initially, the production fermentation conditions were set at 37° C.,800 rpm, and 1 vvm (volume of vessel per minute). As the dissolvedoxygen was consumed during the growth time, both agitation and aerationrates were increased accordingly to maintain a minimum dissolved oxygen(D.O.) at 30%. The pH of this medium was consistently self-adjusted at6.5-6.8, optimum for E. coli growth, such that acid and base control ofthe medium pH became unnecessary. Approximately four hours followinginoculation into the production medium, cell density reach an O.D.₆₀₀ of10 when L-arabinose (50 g) was added to induce the synthesis of cecropinfusion protein. To further ensure the stability of the expressionvector, 1 g of ampicillin was supplemented to the broth at an O.D.₆₀₀ of20.

Both alpha-TGF and cecropin-araB fusion proteins were localized only inthe insoluble fraction of the E. coli cells. Microscopic examinationshowed insoluble inclusion bodies were formed inside the cells one hourfollowing induction and were stably maintained throughout thefermentation. More than 95% of the cells contained at least oneinclusion body which enlarged rapidly as the cell density keptincreasing. The yield of cell mass was 13.1 g (dry weight) per liter.From the cell mass, approximately 30% was the fusion protein.

EXAMPLE 6 Construction Of E. coli Vector In Which Expression OfaraB-hTGF Fusion Protein Is Under Regulation of S. typhimurium araBPromoter

The araB promoter is positively regulated by the araC gene product.L-arabinose interacts with the araC protein to form an activatorrequired for expression of the araB promoter. A plasmid which containsthe S. typhimurium araB and araC genes was used to construct thearaB-hTGF (human tumor growth factor alpha) expression vector. Thestrategy of plasmid construction is shown in FIG. 8. The final plasmidphTGF5 contains an araB-hTGF fusion which codes for a protein of 548amino acids. E. coli strain MC1061 containing phTGF5 was grown inminimal glycerol (1%) medium supplemented with casein hydrolysate (0.5%)and thiamine (1 ug/ml). When the density of the culture reached anA600=0.2, L-arabinose was added to 1% and incubation continued for fivehours before the culture was harvested. A 55 KDal protein whichrepresents approximately 10% of the total cellular protein was detectedby SDS-PAGE.

EXAMPLE 7 Transcription Terminator 3' Added to the araB-hTGF gene

A transcription terminator is a DNA sequence which causes RNA polymeraseto stop transcription. Placement of a transcription terminator at the 3'end of the araB-hTGF gene will prevent the expression of the undesiredgene product(s) downstream from the transcription terminator. Thestrategy of plasmid construction as shown in FIG. 9. The final plasmidphTGF58 contains an E. coli rrnB gene transcription terminator insertedat the 3'-end of the araB-hTGF gene. When the partially purifiedaraB-hTGF proteins isolated from E. coli strain MC1061 containing eitherphTGF58 or phTGF5 (the parent plasmid) were compared afterelectrophoresis on SDS-polyacrylamide gel, a major contaminant protein,beta-lactamase, was significantly reduced in phTGF58-containing cells.

EXAMPLE 8 The araB Promoter and Calcitonin

A. The human calcitonin gene (hCT)

Calcitonin or thyrocalcitonin (CT) is the name given to the hypocalcemichormone secreted from the thyroid. CT decreases bone resorptiveactivity. CT also acts on kidneys to stimulate increased urinary calciumand phosphate clearance. The rapid release of CT in response to bloodcalcium elevations suggests that the main purpose of CT is to protecthigher animals from acute hypercalcemic episodes. Research in CT isexamining its use in the control of Paget's disease, a problem ofaccelerated bone remodeling.

The sequence for the human gene for calcitonin is as follows (Craig etal., Nature, 295: 345-347 (1982)): ##STR3##

B. Construction of a plasmid carrying an inducible regulon, the araBstructural gene and the hCT Gene

Plasmid p115, its construction shown in FIG. 10, contains the correctcalcitonin gene sequence. The plasmid was digested with endonucleasesMstI and PstI to excise the calcitonin gene, as shown in FIG. 11. Theexcised fragment was ligated to a plasmid carrying the araB structuralgene (plasmid pING1. see FIG. 12).

Plasmid pMH6 (Horwitz et al., Gene, 14: 309-319 (1981)) which containsintact araC and araB was used as starting material for construction ofpING1. The construction scheme is shown in FIG. 12. The plasmid pMH6 wasdigested with restriction endonuclease FcoRI and SalI, which cut in araBand araA gene, respectively, and the 1.9 kb SalI-EcoRI fragment wasreplaced by a 16 base-pair SalI-EcoRI fragment from M13 mp9 (Vieira andMessing, Gene, 19: 259-268 (1982)) to generate pING1. The reading frameof the araB gene was determined by fusion of the lacZ gene to the EcoRIsite in pING1. Since both restriction endonuclease SmaI and MstI createblunt end fragments, the 760 base-pair fragment of pING1 was thenreplaced by a MstI-PstI fragment from plasmid #115 to generate phCT1.Plasmid #115 has the chemically synthesized hCT gene located in theMstI-PstI fragment. The joining of the MstI and SmaI end fused the hCTgene to the araB gene making an araB-hCT protein fusion.

C. Growth of E. coli cells containing phCT1

Transformed E. coli cells containing phCT1 were grown at 37° C., withshaking, to a density of 10⁸ -10⁹ /ml, and the synthesis of thearaB-calcitonin fused protein was induced by the addition of L-arabinose(1% wt/vol) during additional growth for 1-6 hours. The harvested cellswere sonicated (5 sec, 3 times) and the concentration of araB-calcitoninwas assayed by an ELISA procedure using anti-hCT antibodies.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited by thescope of the appended claims.

What is new and intended to be covered by Letters Patent of the UnitedStates is:
 1. A genetic construct which comprises a first geneticsequence coding for a polypeptide which is capable of suppressing thebactericidal effect of the resulting fusion protein towards an otherwisececropin-sensitive bacterium operably linked to a second geneticsequence coding for cecropin.
 2. The genetic construct of claim 1wherein said polypeptide is a leading polypeptide.
 3. The geneticconstruct of claim 1 wherein said genetic sequence coding for saidpolypeptide comprises between about 300 and 5,000 base pairs.
 4. Avehicle replicable in a given host comprising the genetic construct ofclaim
 1. 5. The vehicle of claim 4 which is expressible in a bacterium.6. The vehicle of claim 4 wherein said cecropin is A, B or D.
 7. Thegenetic construct of claim 1 which also includes an inducible promoterin operable linkage therewith.
 8. The genetic construct of any one ofclaims 1 or 7, wherein said genetic sequence coding for cecropin is asynthetic sequence.
 9. The method of claim 8 which further includes astep of cleaving the protein conjugate into cecropin and said differentpolypeptide.
 10. The genetic construct of any one of claims 1 or 7,wherein said cecropin is cecropin A, B or D.
 11. A host transformed withthe genetic construct of any one of claims 1 or
 7. 12. The host of claim11 which is a cecropin-sensitive bacterium.
 13. The host of claim 11wherein said cecropin is A, B or D.
 14. A cecropin-sensitive hosttransformed with a vehicle expressible therein, said vehicle carrying agenetic sequence coding for cecropin operably linked to a geneticsequence coding for a polypeptide capable of suppressing thebactericidal effect of said cecropin towards said host in the resultingexpression fusion product.
 15. A method of producing cecropin bymicrobial techniques which comprises:operably linking a first geneticsequence coding for cecropin with a second genetic sequence coding for adifferent polypeptide to form a fused gene; transforming said fused geneinto a cecropin susceptible host; culturing said transformed host,whereby a protein conjugate non-toxic to said host is produced; andsubsequently recovering cecropin from said protein conjugate.